Project Summary/Abstract The suprachiasmatic nucleus (SCN) in the hypothalamus controls a wide range of circadian rhythms, but the brain circuitry by which it regulates these physiological and behavioral cycles is poorly understood. In the first cycle of this project, we identified the pathways and neurotransmitters that regulate circadian rhythms of wake- sleep, locomotor activity, and aggression. Here we focus on the hypothesis that the SCN controls the circadian rhythm of corticosteroid (CORT) secretion by direct projections to GABAergic neurons in the subparaventricular zone (SPZ), which then innervate both glutamate and GABA neurons in the dorsomedial hypothalamic nucleus (DMH). We hypothesize that the DMH glutamate cells may directly excite paraventricular nucleus (PVH) corticotopin-releasing hormone (CRH) neurons, driving the peak in daily corticosterone prior to awakening. GABA neurons in the DMH, on the other hand, may fire at the same time to inhibit GABAergic neurons around the border of the PVH, which tonically inhibit CRH neurons. We will test this hypothesis with three aims: Aim 1 will examine the effect of deleting the gene for the vesicular GABA transporter (Vgat) in neurons in the SPZ, using an adeno-associated viral vector (AAV) coding for Cre- recombinase in mice with loxP sites flanking exon 2 of the Vgat gene. We expect that injections of the AAV- Cre into the ventral SPZ will diminish or eliminate the daily rhythms of CORT secretion. In Aim 2, we will determine whether the DMH GABA or glutamate cells, or both, are necessary for CORT rhythms. We will use genetically targeted methods in either Vgat-Cre mice, or mice with Cre recombinase under the vesicular glutamate transporter 2 (Vglut2-Cre mice), to either kill Cre-expressing neurons with an AAV coding for Cre- dependent expression of diphtheria toxin A protein, or reversibly silence cells with a Cre-dependent mutated human glycine receptor (hGlyR), that is gated by the antibiotic ivermectin. We will test whether removing either GABA or glutamate cells from the DMH either chronically (with DTA) or acutely (with the hGlyR-ivermectin) prevents circadian rhythms of CORT. Finally, in Aim 3 we will express channelrhodopsin in a Cre-dependent manner either in DMH glutamate or GABA neurons. We will then examine the effect of driving those neurons optogenetically on CORT secretion in the whole animal, and on the post-synaptic potentials recorded from CRH neurons in the hypothalamic slice. We will use confocal microscopy to determine whether there are appositions of channelrhodopsin-labeled DMH glutamate and GABA terminals with PVH CRH or GABA cells, and the slice preparation to test those appositions for synaptic connectivity and neurotransmitter specificity. These experiments will test our hypothesis that the SCNGABA?SPZGABA?DMHglutamate?PVHCRH and SCNGABA?SPZGABA?DMHGABA? periPVHGABA? PVHCRH neuronal circuits regulate the pre-awakening circadian surge of CORT secretion, which is necessary to anticipate and meet the metabolic, physiologic, and cognitive demands of the new day.